Pulmonary Toxicology: Animal Models and Their Utility in the Assessment of Toxic

Inhalants – ENVR430

Nov 16, 18, & 20, 2009

Dan Costa, Sc.D.Director of Air Research USEPAcosta.dan@epa.gov

919-541-2532

Lecture OutlineNovember 16, 2009• Basic lung biology – cross-species perspective• Principles of particle and gas entry into the lungNovember 18, 2009• Basic features of lung toxicity• Acute vs chronic outcomesNovember 20, 2009• Example: PM health issue today

• Witschi H. Chap 15 Toxic Responses of the Respiratory Tract, Casarett & Doull's Toxicology: The Basic Science of Poisons - 6th Ed. (2006).• Costa, DL. Chapter 28 Air Pollution Casarett & Doull's Toxicology: The Basic Science of Poisons - 7th Ed. (2007).• Pope CA, III, Dockery D.: 2006 Critical Review - Health Effects of Fine Particulate Air Pollution: Lines that Connect. JAWMA 56: 709-748, 2006.•Ghio AJ, Devlin RB. Inflammatory lung injury after bronchial instillation of air pollution particles. Am J Respir CritCare Med. Aug 15;164(4):704-8, 2001.• Dye J. et al. Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents. Environ Health Perspect. 109 Suppl 3:395-403, 2001.

Leading Causes of Death in the

U.S. 2003

Accidents: Leading Cause of death <34 yrs of age

Distribution of Asthma in the U.S. Population

The Challenge of Air Pollution Health Sciences

• Exposures to air pollution are inherently “mixtures”• Exposures have temporal and spatial variability• Health-relevant airborne concentrations of most individual

pollutants are quite low - exception is ozone.• In most cases, the gradient of exposure concentrations from

those most exposed to least is small• Impact of confounding factors (e.g., tobacco smoke) is

substantial• The disease outcomes are not uniquely related to air

pollution• Host-pollution interaction may be critical (i.e., susceptibility)

HUMAN STUDIES: EPIDEMIOLOGY & CLINICAL STUDIES

• Should be used whenever possible– Right species– Exposures most relevant – real world

• Limitations of epidemiology – Exposure data are often weak – dosimetry even weaker / assumed– Difficult to establish cause-effect relationships:

• Confounding factors – cigarette smoke; diet; occupational• Effects of various pollutants may be similar• Sometimes the interval between exposure and effect is long

• Controlled Human Studies– Right species but ethical limitations – Study groups may not reflect the demographics– Controlled exposure may need extrapolation

LABORATORY ANIMAL STUDIES: ROLE IN INHALATION TOXICITY ASSESSMENTS

– Defined Subjects: Species; Strain; Age; Sex; Health Status– Control Exposure Conditions

• Specific Compound or Mixture• Well Characterized Atmosphere

– Concentration– Particle Size Distribution– Temperature & Humidity

– Allow Assessment of…• Exposure-dose Relationships (Toxicokinetics)• Exposure (Dose)-Response Relationships (Pharmacokinetics)

– Use of Multiple Species Increases Confidence in Extrapolation

IN VITRO STUDIES:ROLE IN INHALATION TOXICITY ASSESSMENTS

• Provides Basic Mechanistic Information Needed For Extrapolation and Fundamental Understanding – Isolated Perfused Lung or Heart

• Metabolism • Evaluation of Function

– Isolated Cells or Tissues (examples)• Epithelium – function, cytotoxicity, intracellular processes• Alveolar Macrophages - Phagocytic Capacity, Cytotoxicity

– Screening potential – “Toxicology for the 21st Century” (NAS)

The Lung is a Multifunctional Organ• Well-designed for its primary function (O2 CO2)

• All cardiac output passes through the lungs• Primary target for anything in inhaled air

• Maintains blood pH• Exocrine functions –

angiotension, biogenic amines

• Metabolic functions – P450’s• Excretory function –

CO, NH3, organic vapors

The Respiratory System

Weibel et al., 1981

Design Parameters of the Mammalian Lung

Airway surface ~5% total SA

Air-blood barrier ~1m

Comparative Nasal Airway Structure and Function

0.4816-19Volume(cm3)

NasalOronasalOronasalBreathing

Large50%

Moderate20-30%

Small<10%

OlfactoryEpithelialSurface Area

ComplexSimpleSimpleTurbinateAnatomy

RatMonkeyHuman

Harkema et al., The Nose Revisited: A Brief Review of the Comparative Structure,Function, and Toxicologic Pathology of the Nasal Epithelium Tox. Pathology, 34:252–269, 2006

Ventilation• Divisions of lung volume• Tidal Volume (Vt)• Frequency of breathing (f)• Vt x f = Minute Ventilation (VE)

• Transpulmonary pressure (PL)• Computation: Lung Resistance (RL) and Lung

Compliance (Cdyn)

28U.S. Environmental Protection AgencyOffice of Research and Development

PM is a complex mixture of solid, semi-volatile and aqueous materials of various sizes found in the air.

Mineral Fiber Natural Fiber

PollenAnthropogenic

Ultrafine PM (nano)

2.4 million = 1

Particulate Matter

29U.S. Environmental Protection AgencyOffice of Research and Development

NucleationMode

Ultrafine

AccumulationMode

CoarseMode

Fine (PM 2.5) 2.5

PM 10

Los AngelesEastern USMineralsSulfateAmmoniumNitrate

Organic CarbonElemental Carbon

Unknown

Airborne PM is itself a

complex mix of size and chemistry

Gases

Numbers and Surface Area of Different Sizes of Particles of Unit Density at a Mass Concentration of 10 µg/m3

241.22.5

60191.0

1201530.5

60019,1000.1

30162,400,0000.02

Particles Surface Area (µm2/cm3 )

Particle Number (per cm3)

Particle Diameter (µm)

CIIT Model PM Deposition Predictions

Asgharian, et al., 1999Asgharian, et al., 1999

Human Rat

Gases & Vapors

• Gases exist in gas phase at room temperature

• Vapors can coexist as gas and/or liquid at room temperature (have lower vapor pressure)

• Diffusion is a major factor in dispersion in the air

The higher the blood/air partition coefficient, the less desorbed on the ensuing breath –hence accumulation of the vapor by the body (blood and fat stores)

ASTM – E981-84 Mouse Irritancy Bioassay

Assesses the irritant potency of sensory irritants and segregates these responses from pulmonary irritants by examining changes in breathing frequency

Chronic Diseases• Chronic exposures – tobacco smoke (MS & ETS);

outdoor and indoor pollutants• Episodic (often chronic) – occupational (coal, silica

miners); air pollution• Single exposure w/ chronic outcome – accidental

releases (MIC – Bhopal India 1980); occupational; avocation (hobbies)

• Idiopathic disease (unknown origins)• Gene – Environment Interations - (1-AT; GSTM-1)

Emphysema (COPD)Emphysema (COPD)

Emphysema: Loss of Elastic Recoil

Costa, 1985

Functional Outcomes

Compliance Changes with Disease

Volume and Diffusion Changes with Disease

London's "killer smog" of 1952 was so thick that busses had to be escorted by men walking alongside with lanterns.This photo was taken around 10:30 AM.

The Problem: 20th Century Perspective

4-8000 deaths

Donora, PA – 1948

20-50 deaths

London Smog 1873

Houses of Parliament, Claude Monet

Ambient PM Derive from Diverse Sources

AgricultureFine particles can be emitted directly

or formed in the air from gases.

Power Plants

Africa

10/30/03 10:27:18 AM

http://www.firedetect.ssd.nesdis.noaa.gov/viewer.htm

General PM Sources and Composition

PM Composition Varies by Size Mode

““ClassicalClassical”” Air PollutionAir Pollution

Reducing SmogReducing Smog -- Acrid, smoky emissions of coal & fossil Acrid, smoky emissions of coal & fossil fuel combustion: Industry and domestic heatingfuel combustion: Industry and domestic heating•• Particulate matter (PM) Particulate matter (PM) -- complex soot; acidic & metal sulfates (SOcomplex soot; acidic & metal sulfates (SO44

==))•• Sulfur dioxide (SOSulfur dioxide (SO22 ))•• Carbon monoxide (CO)Carbon monoxide (CO)

Birmingham – 1972New York City – 1966

““ClassicalClassical”” Air PollutionAir Pollution

Oxidant SmogOxidant Smog -- EyeEye--irritating, haze of sunny suburbanized irritating, haze of sunny suburbanized cities: Automobile emissionscities: Automobile emissions•• Nitrogen oxides (NONitrogen oxides (NOxx))•• Volatile organic chemicals (Volatile organic chemicals (VOCsVOCs))•• Ozone (OOzone (O33) ) -- photochemical reaction product of NOphotochemical reaction product of NOxx and and VOCsVOCs•• Partially combusted organics (Partially combusted organics (PICsPICs) ) –– often mixed with often mixed with VOCsVOCs and Rx productsand Rx products•• Carbon monoxide (CO)Carbon monoxide (CO)

The Air of our Parents:The US Air Quality Management Program has come a long way

The Modern Dimensions of Air Pollution

• Most urban and suburban environments have oxidizing atmospheres comprised of ozone, primary and secondary particles, and a varying mix of copollutants– Regional pollutants (long range transport) – power industry– Transportation – auto, diesel – Fugitive and accidental releases – VOCs, PAHs, metal cpds. etc.

• Indoor Air exposure is ‘stealth’ and often underappreciated– Penetration of outdoor pollutants– Environmental tobacco smoke– Indoor sources – NOx, VOCs, – Biologicals – molds, dust and cockroach mites etc.

An International Problem

WHO (2002) estimates 2M premature deaths / year

Industrialized nations – show obvious improvementsAdvanced TechnologiesPublic educationGrowing Wealth

Developing nations - urbanization ►deterioration of AQ Population growthUncontrolled IndustrializationEnergy consumptionQuest for rapid wealth

Delhi, 2000

Beijing, 2001

PM Problems Persist Even in the U.S

Atlanta, 2003

75U.S. Environmental Protection AgencyOffice of Research and Development

Size mattersDockery, et al., 1993

Susceptibility

Schwartz et al., 1992

Life-Shortening

In 1997, the PM Epidemiology Was

Compelling…(but with many uncertainties)

Regarding Morbidity…

Rolling Stone Magazine

Science March 25, 2005 (Vol 307) pp.1858-1861

What is it about PM that Makes it a Significant Health Hazard?

• Is it the size and/or composition of PM?• How does PM lead to mortality?• Are there chronic effects?• Who is at risk?• Are there host factors? • Is there a source hierarchy?• Do co-pollutants have a role?• Are there other environmental factors?

HealthyAnimal

DiseasedAnimal

HealthyHuman

DiseasedHuman

Toxicological Paradigm for Use of Susceptible Disease Models

f’(x)f(x)

g(x)

g’(x)

• The human population is heterogeneous, hosting a spectrum of susceptibility factors: gene-environment (including epigenetic); disease; age; life-style…

• New tools open many possibilities

• Classical toxicology has largely focused on relatively young, homogeneous, healthy lab animals. Expanded use of genetic and other models…

• Studies in animal models offer tools for controlled investigation of specific “susceptibility” factors and has both acute and chronic applicability.

EpidemiologyAnimal

Toxicology

Mechanisms

Acidic particles

Transition metals

Organics

Hydrogen peroxide

Ultrafines

Bioaerosols

Dose distribution

Proposed Attribute “Hypotheses”

Sources of PM10 Pollution in the Utah Valley (1985-87)Geneva Steel 82% of industrial emissions when operating

47- 80% of total emissionsWood Burning 16%Road Dust 11%Diesel Fuel 7%Oil Combustion 7%

PM10

(g/

m3 )

0

25

50

75

100

125

150

1985 1986 1987 1988

PM10 Concentrations, Lindon Site

Steel MillClosed

PM10 Levels Correlate with Hospital Admissions for Pneumonia & Pleurisy; Bronchiolitis & Asthma

0

10

20

30

40

50

60

7080

1985 1986 1987 1988

Monthly Bronchiolitis & Asthma Hospital Admissions: All Ages

Steel MillClosed

Pope, Am J Public Health 79:623, 1989

Utah Valley filter extracts

40 mL

Pooled and Lyophilized

Aqueous extracts

Utah Valley filter extract metal analysis

1986 1987 19880.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

CopperZincIronLeadStrontiumArsenicNickelManganeseVanadium

Met

al (m

g) p

rese

nt in

filte

r ext

ract

s Utah Valley extract (12 TSP filters/year)

Dye et al, EHP 2001

Effects on Lung Permeability

1986 1987 19880

50

100

150

200

250

300 Saline(contralateral)Extract

Saline 1986 1987 19880

100

200

300

400

500

Prot

ein

(µg/

mL

BA

L fl

uid)

Rats Humans

Dye et al, EHP 2001 Ghio et al, 2001

(BAL Total Protein)

Prot

ein

(µg/

mL

BA

L fl

uid)Saline

Extract

0

100

200

300

400

500 g/ml250 g/ml100 g/ml0 g/ml

IL-6

(pg/

wel

l)Frampton et al., 1999

198619871988

Proinflammatory Marker

GeneticsMonogenic/polygenic-Species/strain-Gender

Environmental -Exposures-Infections-Nutrition

AgeDisease

Susceptibility – “No longer data outliers”

• Young versus old – excesses at both extremes• Asthma & other preexisting cardiopulmonary disease• Preexisting lung inflammation – infection• Diabetics – systemic inflammation / oxidant load• Genetics - gene-environment interactions (e.g.GST-M1)

Is there a common factor(s)?• Red-Ox imbalances• Loss of compensation / recoverability• Interactions with dosimetry• Multiple factors

Exposure Dose of an Air Pollutant

Homeostasis

Effect

Injury

Clinical Effect

Leve

l of B

iom

arke

r

Severe Effect

How Do We Describe Susceptibility?

Rodent Models of Cardiopulmonary Diseases

• Bronchitis / Emphysema / Fibrosis• Systemic hypertension• Aging / Cardiomyopathy• Allergic asthma• Pulmonary vasculitis / hypertension• Bacterial / viral infections• Genetic and transgenic disabilities

Bronchitis Models

• 200 ppm SO2 6 h/d, 5 d/wk, 6 wks LPS (Gordon & Harkema,’94)

Bronchitis

SO2

Sprague Dawley RatAir

Smith et al., 2002

Airway Cell Metaplasia in SH Rats Following 8-week ETS Exposure

(70-80 mg/m3 6 hrs/day 3 d/wk, 8 wks)

PM Deposition in the Bronchitis

Sweeney et al., 1995

NORMAL

RAT

SO2 BRONCHITIS

HUMAN

Bennett et al., personal communication

BRONCHITIS

Clarke et al.,1999

Impact of CAPS (PM2.5) in Bronchitic Rats

0100

300

500

700

900

Air CAPs Air

BA

LF P

rote

in (µ

g/m

l)

#~600ug/m 3 x 2 days

CAPs

Kodavanti et al., 2000

0

500

Air CAPs Air

BA

LF P

rote

in (µ

g/m

l)

#~500ug/m 3 x 3 days

CAPs

1500

2500

3500

4500

Boston RTP

PM Pulmonary-Cardiac Interactions

PM Exposure

DepositionClearance & ∆PFTs

VentilationCNS

InflammationEicosanoidsCyto/ChemokinesGrowth Factors Reactive O2 & N2

Proteases

PM Dissolution

Bio/Chemical Interactions (endotoxin, metals, PAH’s, reactive O2 & N2)

Chronic Impact Lung Remodeling Cancer

Allergenic / Immune Rxs

A “New” Dimension to Air Pollution

(Pope, 2000)

ELECTROCARDIOGRAPHIC ANALYSIS

SINGLE ELECTROCARDIOGRAPHIC WAVEFORMSINGLE ELECTROCARDIOGRAPHIC WAVEFORM

ADULT RATADULT RATHUMANHUMAN

R-aT

Lo Mid High

SO2 0.05 0.25 1.25 ppm

NO 0.05 0.25 1.25 ppm

NO2 0.04 0.20 1.00 ppm

CO 7.2 36.0 180.0 ppm

CH4 2.7 14.0 67.5 ppm

(NH4) 2SO4 ~25 ~125 ~625 ug/m3

Mid High

Normal rats (30wk) 2/20 1/20

Thrombogenic rats (16wk) 6/10 6/10

Hypertensive rats (32wk) 9/12 11/11

Anemic rats (16wk) 1/8 0/8

Hartroft et al., The Institute of Elect and Electronics Engineers, USA Publication #75CH1004-134-5:1, 1976

Susceptible Rats and Complex Atmospheres

Inhaled ROFA (15 mg/m3 6 hr/d, 3d)-induced hemolysis and BAL RBC’s

SH Rats Exhibit Greater Lung Injury Following PM Exposure

WKY-AIR

WKY-ROFA

SH-AIR

SH-ROFA

Kodavanti et al., 2000

772 MI patients

OR = 1.69 (1.13-2.34) for a 20 g/m3 increment in 24-hour PM2.5

Peters et al., 2001

-0.6-0.4-0.20.00.20.40.6

Healthy Subjects

Compromised Subjects

HF LF HF LF

Heart Rate Variability

Liao et al., 2000

Evidence that PM affects the Cardiovascular System

Watkinson et al., 1998

ECG Abnormalities and death in fly ash exposed hypertensive rats

Important Generic Questions to Ask of Animal Models

Given: The pathophysiology of human disease is variable….

At what pathophysiologic stage does susceptibility become evident? Is the response coherent with that of the human - esp. mechanistically? What are the implications of a genetically homogeneous host on the

response being studied? Do host attributes interact? (lung & heart; age; systemic factors)

Does the underlying responsiveness reflect altered dosimetry, a unique mechanism for the condition, or a loss of functional reserve?

Fine-Particulate Air Pollution and Life Expectancy in the United StatesPope, Ezzati, & Dockery, N Engl. J.Med. 2009;360:376-86

How do we know PM reductions actually have public health benefit?

Accountability

0 5 1 0 1 5 2 0 2 5 3 0 3 50 .7

0 .8

0 .9

1

1 .1

1 .2

1 .3

P

T W

HL

S

T

W

H

L

S

W

L

HS

TW

LH

S

T

Annual Average PM Concentration, µg/m3

Mor

talit

y R

isk

Rel

ativ

e to

Por

tage

, WI

Follow-up to the Harvard Six Cities Study Indicates Reduced Air Pollution Results in

Lowered Health Risks.

Laden et al, 2006

0

1

2

3

4

5

0 2 4 6 8 10 12 14 16Reduction inPM2.5 (g/m3)

L

ife E

xpec

tanc

yLife Expectancy vs PM2.5, 1980-2000

Slope +.61 yr/10 g/m3

71

72

73

74

75

76

77

78

79

80

81

5 10 15 20 25 30 35PM2.5 (g/m3)

Life

Exp

ecta

ncy

Slope -2.1 yr/10 g/m3

EPA NAAQS

Life Expectancy vs PM2.5, 1997-2001

106U.S. Environmental Protection AgencyOffice of Research and Development

• What does this finding mean relative to other life-expectancy factors?– E.g., relative to the impact of obesity?

PM Slope +.61 yr/10 g/m3

Obesity

Olshansky et al., NEJM 352:11 (2005)

Pope et al., NEJM 360:376 (2009)

What do we understand about PM risk and biologic plausibility?

• Attributes of PM composition/size (perhaps in concert with copollutants?) drives the health responses.

• “Dose” is fundamental to response and often goes un-appreciated in the context of susceptibility.

• Host attributes may be the primary determinant of risk.

• Impacts may not necessarily be greater than in healthy individuals, but may be additive with pre-existing factors or simply may overwhelm reserve and compensatory capacities.

• Accountability – science is showing that regulatory approaches are making a difference; will there be a “climate penalty”?